Chapter 2: Processing Flashcards
What is the most common dehydrating agent?
Alcohol.
During microtomy, it is noted that many of the tissues are very hard and shrunken. What happened?
The infiltrating paraffin was too hot.
What is the appropriate temperature range for infiltrating paraffin during processing?
No more than 2-4C above the melting point of paraffin.
Decalcification of small specimens can be achieved by fixation in:
Zenker. Because it contains acetic acid, it would be ideal for bone marrows and small needle cores.
What is a major disadvantage of aliphatic clearing agents?
They are incompatible with mounting media.
What is an aliphatic clearing agent?
A xylene substitute.
What will cause sensitization with prolonged use?
Limonene. It can also cause allergic reactions in some individuals.
Too speed up the lab’s processing of all surgical tissue, the temperature of all fixation, dehydrating and clearing steps has been set to 45C. What will this result in?
Microchatter due to the removal of bound water from the tissue.
What does limonene function as?
Limonene is a xylene substitute so it would be a clearing agent.
What is one advantage of aliphatic hydrocarbons?
They are low in toxicity and sensitization (sensitivity).
What is one disadvantage of using heat at all stations in the tissue processor?
Some tissues will become hard.
Dehydration refers to the removal of:
Water.
What kind of decalcification process may cause damage to the tissue?
Electrolytic.
Is chloroform combustible?
No, but it is very toxic.
What groups of reagents are used for dehydration?
Dioxane, methanol and ethanol.
When is EDTA NOT a suitable choice when decalcifying bonemarrow specimens?
When a diagnosis is required within 48 hours.
A clearing agent MUST be miscible with:
Dehydrants and infiltrating media (the steps before and after it).
Why should the alcohols be changed routinely on the tissue processor?
The alcohols absorb water from the tissues and become diluted over time.
After decalcification, what should you do with the tissue?
It should be washed in water to halt the reaction.
What is a common chelating agent used for decalcification?
EDTA.
How does ethanol function during processing?
It functions as a dehydrant.
Butyl alcohol is used as a dehydrant for what kind of tissue?
Plant tissue.
The processing step that assures that alcohol is removed from the tissue is the:
Clearing step.
Dehydrating tissue in graded alcohols instead of only absolute is superior because:
Tissue shrinkage is minimized when graded alcohols are used.
For best support during microtomy, decalcified bone specimens which have been exposed to paraffin wax should be embedded in:
“hard” paraffin wax.
What is the best method of preparing tissue for enzyme demonstration?
Unfixed frozen sections. Heat will speed up the enzymatic processes (decomposition).
How can glycol methacrylate function in a processing cycle?
It can function as a Infiltration medium that is converted to a solid by polymerization.
What infiltration medium should be used to preserve fats?
Carbowax because it does not require the use of solvents that dissolve lipids.
How does methanol function in a processing cycle?
It functions as a dehydrant.
What is the most effective method for rapidly freezing tissue?
Liquid nitrogen and isopentane.
Does the processing schedule have any impact on the time that a bone marrow specimen needs to be decalcified?
No.
When tissues have been fixed in Carnoy fluid, which processing solution should it be placed in first.
The tissue should be placed in either 95% or absolute alcohol.
Tissue must be dehydrated before placing it in:
Epoxy resin.
In order to process tissue faster, what could be done easily and will still yield a good result?
Adding agitation to each step during processing.
What is the ideal thickness of tissue prior to decalcification?
3-4mm.
Paraffin processing is contraindicated for the demonstration of:
Enzymes because the heat from paraffin processing will destroy them.
What is miscible with water, alcohol, hydrocarbons and paraffin?
Dioxane, it is a universal solvent.
Reprocessing tissue may lead to false negative IHC results because:
Repeated, heated paraffin exposure alters the epitope sites in the tissue that may lead to a false negative result.
What is the LEAST desirable method to check for the decalcification endpoint?
Physical methods because it could poke a hole in the tissue and produce an artefact.
What will prolonged dehydration in higher grades of alcohol do to a specimen?
The tissue will become hard.
For adequate clearing in xylene during routine processing, tissue should have a maximum thickness of:
3-4mm.
What is the purpose of using a hydrometer in the histopath lab?
Since they measure the specific gravity, they can be used to check the concentration in our alcohol reagents.
Processing of delicate tissues (ex. embryonic tissue), should be started in what concentration of alcohol?
30% because higher concentrations of alcohol can cause distortion of the tissue.
What will cause hazy, blue nuclear staining?
It is caused my improper heat applied during the alcohol (dehydrating) and xylene (clearing) steps.
The effect of overdecalcification is most noticeable in the staining of:
Nuclei, because over exposure to decalcification acids increases nuclear basophillia.
What is considered the BEST dehydrant?
Ethanol.
Are electrolytic procedures used for the checking of decalcification endpoints.
No.
Acid solutions soften bone tissue by removing what salt?
Calcium salts so that the bone will be soft enough to be cut during microtomy.
What should the clearing reagent be miscible with?
It should be miscible with the reagent before and after it, so the dehydrant and paraffin.
If the clearing agent is cloudy, it is most likely contaminated with:
Water because it reacts with the hydrocarbons in the clearing reagents.
What is dioxane’s main functionality?
It is a universal solvent in that it can be used as both a dehydrant and a clearant.
What gas is released during decalcification?
Carbon dioxide.
What is an advantage of adding eosin or phloxine to processing alcohols?
It will make small needle cores more visible during embedding and microtomy.
T/F The fixative used on a specimen will NOT have any impact on the degree of paraffin infiltration.
True.
How does xylene function during processing?
It functions as the most common hydrocarbon clearing agent.
What method of checking decalcification endpoints is the most likely to produce an artefact?
Physical methods, you could poke a hole in the tissue.
What is the best way to hold small pieces of tissue together in place for processing.
The use of Agar on the tissue ex. cell blocks.
Can radiography be used to decalcify specimens?
No Radiography is only really used to check the decalcification endpoint.
Which infiltration reagent is most miscible with hydrocarbons.
Paraffin wax, it is miscible with xylene and it not water soluble.
What is the process of saturating tissue with embedding medium called?
Infiltration.
What is the primary function of toluene?
It is an uncommonly used clearing reagent.
What is the BEST substitute for ethanol in processing?
Isopropanol is a good substitute for processing but NOT for staining (we used this at Kaiser).
How is microchatter caused?
Tissue drying from overdehydration.
What is the most important thing to remember while using essential oils as a clearing agent?
It is necessary to remove the oil with xylene prior to infiltration. We use this with the peanut oil/ xylene combination during the Fite stain.
Freezing tissue slowly prior to sectioning will most likely:
Yield sections showing tissue disruption caused by large ice crystals. This is most prevalent in muscle biopsies.
After H&E staining, a decalcified tissue shows some areas of very dark purple staining. What happened?
The bone section was not put in the decalcification solution long enough, those are calcium deposits.
Why is determining the endpoint of decalcification important?
Overdecalcification will result in the destruction of cellular structure. It is better to undercook than overcook.
A melting point of paraffin of 55C is used for impregnation and embedding. What should the containers of paraffin be regulated at to produce this temperature?
58C because the temperature should not exceed 4C more than the melting point of paraffin.
What is the melting point of paraffin?
55C.
When calculating the time for a perfect infiltration of paraffin into tissue, the time needed depends on the:
Thickness and texture of the tissue.
What is the step that must be completed before dehydration?
Fixation.
T/F The rapid removal of fixative is a factor that should be taken into account when choosing a suitable clearing agent.
False. Clearing agents do not remove the fixative, they come after the dehydrant and are not miscible with fixatives.
What class of chemicals does paraffin belong to?
Long chain hydrocarbons.
For the BEST results before decalcification in acids, a buffered formalin fixed specimen should be:
Washed in water to remove phosphates so they will not react with the decalcification acids.
When EDTA is used as a decalcifier, the recommended pH of that solution is:
7.0-7.4
When does a bone NOT have to be fixed before decalcification?
When the decalcifier is combined with a fixative into one solution.
T/F A properly decalcified specimen will show the retention of calcium in the tissue.
False, removing the calcium is the entire point of decalcification.
Although benzene is very rarely used as a clearing agent, one advantage of using it is:
Benzene is very volatile and evaporates very quickly from paraffin at it’s melting point so the wax will not need to be changed on the processor.
What is the slowest chemical decalcification reagent?
EDTA.
In order to pour decalcification reagents down the drain, first they must be:
Neutralized with 1% sodium bicarbonate.
When a lab uses tetrahydrofuran in processing, the tissue that will be most likely to show distortion is:
Delicate tissues such as needle cores and cell blocks.
When tissue is fixed in a primary formaldehyde fixative and then placed directly into HCl for decalcification, what is the expected outcome?
Bis-chloromethyl, a powerful carcinogen, is formed.
What decalcification method requires the LEAST frequent changing of the solution?
Ion exchange because the ammonium ions in the resin are exchanged for the calcium which keeps the acid free from calcium ion buildup.
Ethyl alcohols used in processing are not fit for human consumption due to the addition of:
Methanol. Since it is non-consummable, the alcohol is not taxed.
What is the most important thing to remember before surface decaling a block?
Remember to face in the block before treating with decal solution.
What is the recommended schedule for monitoring decalcification completeness?
Dependent on the strength of decal reagent used.
Microwave processing is most useful for what specimen type?
Small biopsies because they can be processed quickly and well with the microwave.
When employing microwave fixation, the tissue sample should be:
Fixed before processing.
The biggest benefit of microwave processing is:
The decreased time for completion.
During paraffin processing, how many changes of paraffin wax are recommended for adequate infiltration?
3.
When a fixed liver biopsy is received in the lab for rapid processing, it is recommended that:
Fixative solutions be skipped because the tissue has already been fixed.
To assist the embedder in identifying small and colorless tissue samples, it is recommended that:
A small amount of eosin or phloxine is added to the final dehydrating step.
Dehydrating during tissue processing occurs by repeated dilution of:
Hydrophilic property of alcohol because it draws water out of the tissues.
When a block of UNDER-calcified tissue is being cut and a section cannot be obtained, what is the best course of action?
Place the faced surface of the block in 5% HCl.
One purpose of the additives such as plastics that are in some paraffin wax is to:
Increase hardness to embed bone tissue.
What is “hard” paraffin?
Paraffin that has some plastic added to it. It is commonly used to embed bone tissue.
The method most commonly used with cytology preparation of a scant nongyn fluid specimen is to:
Use the cytocentrifuge before making a cell block.
In order to process faster and still yield similar results, what is recommended BESIDES agitation?
Adding vacuum to each reagent step because it will increase the infiltration of all processing fluids.
How often should the reagents be changed on the processor?
It depends on the amount of use, degradation depends on the number of blocks.
When small biopsies and large tissue specimens are processed together with a processing schedule that will adequately process the larger specimens, what will happen to the small biopsies?
They will become overcooked, i.e. hard and overly dry.
To assure adequate and appropriate tissue processing, in which step should heat be applied?
Infiltration ONLY because applying heat to the other steps will generally result in hard, brittle tissue.
How do resins work for EM processing?
They harden by crystallization.
A tech in the EM lab has developed dermatitis. What happened and how can this be prevented?
PPE needs to be worn during all steps in processing.
Smudgy nuclei and variations in nuclear staining are noted on routinely processed, formalin fixed biopsy specimens. What happened?
Incomplete dehydration. The alcohols need to be looked at.
Processed tissue that was fixed in zinc-formalin is very hard and brittle with microchatter in stained sections. What happened?
The tissue has been overhardened and over processed, the processing schedule needs to be changed.
A tech in the EM lab has developed dermatitis. What chemically were they specifically exposed to?
Epoxy resins.
When placed in a solution whos refractive index is similar to the refractive index of tissue proteins, what happens to the tissue?
The tissue becomes translucent because it becomes the same color as the background.
Microscopic review of H&E stained sections of a colon biopsy shows very uneven staining of the tissue and poor nuclear detail. What is the most LIKELY cause?
The clearing agent was contaminated with water so the clearing didn’t happen effectively.
Xylene, toluene and benzene belong to what class of chemicals?
Hydrocarbons.
To help maintain morphology in formalin fixed tissue prior to freezing, fixed tissue may be placed in a solution of:
30% sucrose.
T/F the viscosity of reagents is DIRECTLY related to the rate of clearing.
True. The less viscous, the faster it will clear.
The penetration of any solution into tissue is increased as what is increased?
Temperature, it is a direct relationship.
To BEST demonstrate muscle enzymes, which freezing method should be used?
Isopentane chilled to -150C.
Polyethylene glycol is employed as the embedding medium for the preservation of:
Lipids.
What is the advantage of increasing the temperature in the tissue processing chamber?
The flow of reagents into the tissue will improve.
When processing with water soluble wax, tissue is fixed, washed with water and:
Infiltrated only. Tissues processed with water soluble wax do not require dehydration or clearing.
What kind of infiltrating media should be used when the tissue must be embedded in a medium that will tolerate a small amount of water?
Glycol methacrylate.
If a lab ran out of ethanol, what would be a suitable substitute?
Isopropanol (Kaiser does this).
The practice of “double embedding” includes the use of which two compounds?
Agar and then paraffin, usually used with cell blocks.
High resolution light microscopy is needed on a lymph node biopsy. To achieve the BEST results, which embedding medium should be used?
Glycol methacrylate.
A section of kidney is fixed in phosphate-buffered formalin and then routinely processed with 95% alcohol, absolute alcohol, xylene and then paraffin. When sectioned, vertical knife lines are observed in the sections and still remain even after a new blade is used. What happened?
Precipitated phosphate remains in the tissue causing micro calcifications. The tissue should have been adequately washed prior to processing.
What is a major disadvantage of using Carbowax?
Carbowax is water soluble so the tissue sections cannot be floated on a water bath.
What would happen if the paraffin was infiltrated at 70C?
The tissue antigens will be denatured.
An oil red O stain has been requested on a friable specimen that must be embedded for sectioning. What embedding medium should be used?
Water soluble wax because the paraffin would dissolve the lipids that the oil red O would showcase.
Microwave processing generally requires the use of what reagent?
Isopropanol because it is miscible with paraffin.
While performing the daily temp check of the embedding center, it is noted that the paraffin reservoir is measured to be 12C above the melting point of paraffin. What should you do about it?
Recheck the thermostat setting.
A method of decalcification is needed for research bone specimen on which oxidative enzyme stains will be ordered. What decalcifying method should be used?
Chelating agent such as EDTA because it is the most “gentle”.
In the electrolytic method of decalcification, the specimen to be decalcified is attached to the anode. Why is this?
The calcium ions will be attracted to the cathode.
In order to maintain the BEST morphology in tissue samples to be decalcified, it is imperative that:
The specimen is fixed prior to decalcification.
After H&E staining, it is noted that the tissue processed overnight all lack chromatin definition in the nuclei and there is generally uneven staining. What happened?
Water remained in the tissue during infiltration.
In order to ensure that very scant pieces of tissue/ cell blocks are not lost during processing, what should be done?
The tissue should be placed in liquid agar and allowed to solidify prior to processing.
What is the appropriate method for processing a body fluid with spontaneous clots?
Squeeze out excess fluid from the clot and wrap it in lens paper prior to processing.
There have been problems with tissue autoflourescence in situ-hybridization (FISH) procedures. What could be a possible cause of this?
Eosin is in the dehydrant and eosin will cause FISH interference.
To avoid tissue autoflourescence, a good colorant for the tissue dehydrating section would be:
Methylene blue.
What is the main disadvantage of using dioxane in tissue processing?
Dioxane is highly toxic.
The main objective of using xylene as a clearing agent is:
Xylene hardens tissue and is a clearant.
What is the main characteristic of “soft” paraffin (no plastics)?
It is the most useful when thick sections are required.
When used for clearing, cedarwood oil must be followed by:
Xylene, similar to the Peanut oil and xylene for the Fite stain.
Why is paraffin cooled as rapidly as possible after embedding?
Prevent the formation of large paraffin crystals.
T/F: Specimens for EM are embedded in glycol methacrylate.
False, EM specimens are embedded in epoxy resin.
T/F: Sections processed by the celloidin method show less shrinkage than when processed by the paraffin technique.
True because no heat is involved.
T/F: Toluene is the preferred reagent for microwave processing.
False. Microwave processing uses only ethyl alcohol, isopropyl alcohol and paraffin.
T/F: Carbowax is the blend of liquid polyethylene glycol and wax.
True.
T/F: Xylene and toluene are aromatic hydrocarbons.
True.
T/F: One cause of uneven staining with H&E can be water contamination of the clearing agent on the processor.
True.
T/F: Tissues are usually infiltrated from paraffin directly from an essential oil.
False. Essential oils in general need to be rinsed with xylene prior to infiltration.
T/F: Bone sections should be embedded parallel to the long axis of the block.
False. Microtomy will be easier if the bone section is embedded diagonally in the block.
At microtomy, formalin fixed paraffin embedded tissue kidney sections appear soft and mushy. What happened?
Inadequate dehydration and clearing.
The tissue has loosened from the chuck while frozen sectioning. What happened?
Applying the embedding medium to a warm chuck.
What is the most popular processing medium?
Paraffin.
What is the most common cause of torn and fragmented sections?
Incomplete fixation and poor processing.
What is the most common cause of compressed sections?
A dull blade, too little tilt, cutting too rapidly or paraffin accumulation on the blade.
What causes extreme separation in tissue.
Prolonged flotation on the water bath.
What are the three main categories on IC control?
Absorption, Ag and Negative.
In addition to the heat factor, what is the other important factor in heat-induced epitope retreival?
The composition of the retrieval solution.
The lab has received a report from the QC program concerning the last slides submitted for evaluation. The H&E stains were downgraded because only two shades of eosin could be seen. To get the required three shades, how should the H&E stainer program be changed?
The time in the lower alcohol dilutions should be increased.
In paraffin sections, hepatitis B surface Ag can be demonstrated with which stain?
Orcein.
What nerve cells are predominately found in the CNS?
Astrocytes.
What is the point of contact between the axon of one neuron and the dendrite of another?
A synapse.
Listeria monocytogenes (the rarest form of meningitis) can be BEST demonstrated in a paraffin section with what stain?
The Gram stain.
Immunologic staining can be BEST adapted for localization of surface Ag by electron microscopy after staining with what procedure?
Colloidal gold.
What are the organisms found on gastric mucosa that are the presumptive cause of gastritis (ulcers) and that are stained with silver impregnation?
H. Pylori
What is a good procedure of determining cellularity during procedures, such as FNA?
A toludine wet blue film.
A liver biopsy from a patient with suspected Wilson’s disease shows cirrhosis. A diagnosis could be confirmed by using rubeanic acid to demonstrate the presence of:
Copper, because the accumulation of copper is the cause of Wilson’s disease.
Why will the PAS still stain Coccidioides immitis?
Because the organisms contain carbohydrates.
In IC staining, what is the limitation of polyclonal Ab techniques as opposed to monoclonal Ab techniques?
Polyclonal Ab stain more than one epitope so there is a greater cross-reactivity with similar Ag.
T/F: Sarcomas will react positively with Vimentin.
True.
What is the most widespread mycotic disease in humans?
Candidasis.
What are the two non-neuronal cells most commonly associated with nervous tissue?
Schwann and glial cells.
Microscopic review of H&E liver stains show some blue staining of the cytoplasm. What happened?
There was inadequate fixation of the hematoxylin, liver should never have blue cytoplasm.
What is the primary function of auxochromes in artificial dyes?
They are ionizing groups that give the dye affinity for the tissue.
Where are the chromaffin cells located in the adrenal gland?
Chromaffin is normally found in the cells of the adrenal medulla as dark brown granular material.
What is the refractive index of the resinous mounting media used for stained sections?
1.53-1.54, which is the average refractive index of most tissue.
The Bielschowsky method involved double impregnation of brain sections silver nitrite solution. What is the reducing agent used in this reaction?
Formaldehyde.
T/F: Carbol fuchsin will stain Treponema pallidium.
False, because it doesn’t stain spirochetes.
